System and method for capturing and analyzing cells

ABSTRACT

A system and method for capturing and analyzing a set of cells, comprising: an array including a set of parallel pores, each pore including a chamber including a chamber inlet and a chamber outlet, and configured to hold a single cell, and a pore channel fluidly connected to the chamber outlet; an inlet channel fluidly connected to each chamber inlet of the set of parallel pores; an outlet channel fluidly connected to each pore channel of the set of parallel pores; a set of electrophoresis channels fluidly coupled to the outlet channel, configured to receive a sieving matrix for electrophoretic separation; and a set of electrodes including a first electrode and a second electrode, wherein the set of electrodes is configured to provide an electric field that facilitates electrophoretic analysis of the set of cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/163,185, filed 24 Jan. 2014, which claims the benefit of U.S.Provisional Application No. 61/757,141 filed on 26 Jan. 2013 and U.S.Provisional Application No. 61/757,139 filed on 26 Jan. 2013, all ofwhich are incorporated in their entirety herein by this reference.

TECHNICAL FIELD

This invention relates generally to the cell sorting field, and morespecifically to a new and useful system and method for capturing andanalyzing cells within the cell sorting field.

BACKGROUND

With an increased interest in cell-specific drug testing, diagnosis, andother assays, systems that allow for individual cell isolation,identification, and retrieval are becoming more desirable within thefield of cellular analysis. Furthermore, with the onset of personalizedmedicine, low-cost, high fidelity cellular sorting systems are becominghighly desirable. However, preexisting cell capture systems suffer fromvarious shortcomings that prevent widespread adoption for cell-specifictesting. For example, flow cytometry requires that the cell besimultaneously identified and sorted, and limits cell observation to asingle instance. Flow cytometry fails to allow for multiple analyses ofthe same cell, and does not permit arbitrary cell subpopulation sorting.Conventional microfluidic devices rely on cell-specific antibodies forcell selection, wherein the antibodies that are bound to themicrofluidic device substrate selectively bind to cells expressing thedesired antigen. Conventional microfluidic devices can also fail toallow for subsequent cell removal without cell damage, and only capturethe cells expressing the specific antigen; non-expressing cells, whichcould also be desired, are not captured by these systems. Cellularfilters can separate sample components based on size without significantcell damage, but suffer from clogging and do not allow for specific cellidentification, isolation of individual cells, and retrieval ofidentified individual cells. Other technologies in this field arefurther limited in their ability to allow multiplex assays to beperformed on individual cells, while minimizing sample preparationsteps.

Thus, there is a need in the cell sorting field to create a new anduseful cell system and method for capturing and analyzing cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic representation of an embodiment of a system forcapturing and analyzing cells;

FIG. 1B depicts a variation of an embodiment of a system for capturingand analyzing cells;

FIG. 1C is a perspective view of a variation of the system;

FIGS. 2A, 2B, 2C, 2D, and 2E are schematic representations of a first,second, third, fourth, and fifth pore variation, respectively;

FIG. 3 is a top view of a variation of the system;

FIG. 4 is a top view schematic of a portion of a variation of thesystem;

FIGS. 5A, 5B, and 5C depict variations of an encapsulation module of anembodiment of the system;

FIG. 6 depicts a schematic of a portion of an embodiment of the system;

FIGS. 7A-7D are side views of a first, second, third and fourth opticalelement, respectively;

FIGS. 8A, 8B, and 8C are views of a variation of the cell removal tool;

FIG. 9 depicts a specific example of an embodiment of the system;

FIGS. 10A and 10B are schematic representations of an embodiment of amethod for capturing and analyzing cells;

FIG. 11 depicts a portion of an embodiment of a method for capturing andanalyzing cells;

FIG. 12 depicts a portion of an embodiment of a method for capturing andanalyzing cells;

FIG. 13 depicts a portion of an embodiment of a method for capturing andanalyzing cells, including specific forward and reverse primers forAS-PCR;

FIGS. 14A and 14B depict variations of a method for capturing andanalyzing cells; and

FIG. 15 is a schematic representation of an integrated platform at whichembodiments of the system and/or method can be implemented.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of the preferred embodiments of the inventionis not intended to limit the invention to these preferred embodiments,but rather to enable any person skilled in the art to make and use thisinvention.

1. System

As shown in FIGS. 1A, 1B, and 1C, a system 100 for capturing andanalyzing a set of cells comprises: an array 110 including a set ofpores 112, each pore 111 configured to hold a single cell of the set ofcells; an inlet channel 140 coupled to an inlet of each pore; an outletchannel 150 coupled to an outlet of each pore; a set of electrophoresischannels 160 fluidly coupled to the outlet channel, each electrophoresischannel 161 aligned with a pore of the set of pores; and a set ofelectrodes 170 configured to provide an electric field that facilitateselectrophoretic analysis of the set of cells. In one embodiment, thearray 110 includes a set of pores 112, each pore 111 including a chamber113 including a chamber inlet 114 and a chamber outlet 115 fluidlyconnected to a pore channel 117; the inlet channel 140 is fluidlyconnected to each chamber inlet of the set of pores 112; and the outletchannel 150 is fluidly connected to each the pore channel 117 of the setof pores 112.

The system 100 functions to isolate, capture, and hold cells, morepreferably single cells, at known, addressable locations, and further tofacilitate performance of multiple single-cell assays that can beperformed on individual cells (e.g., rare cells in a biological sample).Once cells are captured in defined locations determined by single cellcapture chambers, a fluidic network of the system 100 can be used toprovide and deliver multiple reagents simultaneously or sequentially toenable a variety of cellular, sub-cellular or molecular reactions to beperformed in each of the single cells. The system 100 can also allowoptical interrogation and detection of events on each of the capturedcells at a single cell level. The system 100 can additionally enableselective release and/or selective removal of one or more of thecaptured cells for further processing and analysis. In some embodiments,the system 100 can confer the benefits of real-time cell tracking,viable cell retrieval, and selective downstream molecular analysis(e.g., electrophoresis), either in the same microfluidic chip oroff-chip. In some embodiments, the system 100 can be used to capturecirculating tumor cells (CTCs) and subpopulations of CTCs, such ascirculating stem cells (CSCs), but can additionally or alternatively beused to capture any other suitable cell of possible interest. The system100 is preferably defined on a chip, more preferably a microfluidicchip, but can alternatively be located on or defined by any suitablesubstrate 120.

The system 100 preferably achieves individual cell capture and retentionwithout antibody coated chambers 113, and preferably maintains theviability of the cells throughout isolation, capture, retention, andremoval. The system 100 preferably additionally minimizes clogging, andcan accomplish this by utilizing suitably sized pores 111 and byleveraging massively parallel flow, such that the cells near a sampleinlet 122 configured to transmit the set of cells toward the arraypreferably experience substantially the same pressure as the cellsdistal the sample inlet 122 while minimizing the total pressuredifferential required to flow liquid at high rates through the system100. The variation in pressure felt by cells at the respective ends ofthe array is preferably less than 50% or 75% of the inlet pressure, butcan alternatively be more or less. The sample flow is preferablysubstantially laminar, but can alternatively have any other suitableflow characteristics. The sample flow path is preferably substantiallyunidirectional, but can alternatively be bi-directional. Cell sortingand viability maintenance can additionally be accomplished bycontrolling the sample flow rate through the system, or through anyother suitable means.

In operation, the system 100 preferably receives a biological sampleincluding the set of cells under positive pressure through the sampleinlet 122, which can be coupled to a fluid channel (e.g., an inletmanifold) coupled to a pump configured to provide the positive pressure.Sample flow through the system 100 can be additionally or alternativelyencouraged by providing negative pressure at an outlet (e.g., at anoutlet manifold coupled to an outlet of the array). Alternatively,actuation pressure can be cycled in a pulse-width modulation fashion orsinusoidal fashion to provide net actuation pressure, either netpositive at the inlet or net negative at the outlet. The samplepreferably flows into the inlet channel 140, through the chambers 113and pore channels 117 to the outlet channel 150, with the set of cellsbeing captured in the chambers 113 for further processing and analysis,and other sample components passing out of the system 100. As such,desired cells of a predetermined size are preferably trapped within thechamber 113 as the sample flows through the pores 111, wherein the porechannel 117 dimensions preferably prevent flow of certain cell sizestherethrough. For example, in the variation of the system 100 configuredto capture CTCs, the chambers 113 are preferably dimensioned larger thana CTC, and the pore channels 117 are preferably dimensioned smaller thanthe CTC (but larger than other undesired components in the biologicalsample, to allow passage of the undesired components. However, thesystem 100 can additionally or alternatively be configured to retain andfacilitate processing or any other suitable particle of interest.

1.1 System—Array

The array 110 functions to capture a set of cells of interest inaddressable, known locations, as shown in FIGS. 1B and 1C, such that theset of cells can be individually identified, processed, and analyzed. Asshown in FIG. 1C, the array 110 includes a set of pores 112, each pore111 including a chamber 113 defining a chamber inlet 114 and a chamberoutlet 115 fluidly connected to a pore channel 117. In embodiments, theinlet channel 140 of the system 100 is preferably fluidly coupled toeach chamber inlet 114 of the set of pores 112; and the outlet channel150 of the system 100 is preferably fluidly coupled to each pore channel117 of the set of pores 112. However, the inlet channel 140 canalternatively be configured to fluidly couple to only a portion ofchamber inlets 114 of the set of pores 112, and/or the outlet channel150 can be configured to fluidly couple to only a portion of the porechannels 117 of the set of pores 112 (e.g., in configurations whereinsome of the pores are coupled in series). Preferably, the array 110 isdefined within a substrate 120, by forming microfluidic elements withinthe substrate 120 (e.g., by etching); however, the array 110 can beformed in any other suitable manner (e.g., by lithography, by molding,by 3D printing, by micromachining, by casting, etc.). The substrate 120can be the substrate described in U.S. Pub. No. 2013/0190212, entitled“Cell Capture System and Method of Use” filed 25-Jul. 2012, which isincorporated herein in its entirety by this reference. In a specificexample, the array 110 is defined within a 4-inch silicon substrateusing a three mask photolithographic process and deep reactive ionetching (DRIE) process to etch microfluidic elements into the siliconsubstrate as a mold. In the specific example, the etched elements arethen transferred to 1 millimeter thick polymethylmethacrylate (PMMA)sheets as a substrate 120 using a hot embossing process, which is thenlaminated with a polymethylmethacrylate (PMMA) laminate to definemicrofluidic pathways. In the specific example, lamination includesutilizing an appropriate roller speed, temperature, pressure, andtension of the laminate to ensure a low level of ingress of laminatematerial into microfluidic structures. The substrate 120 in the specificexample has dimensions of 75 millimeters by 25 millimeters, in order tosubstantially match dimensions of a glass microscope slide. However, thesubstrate 120 can alternatively be any other suitable substrate 120. Invariations of the specific example, and/or for other variations of thearray 110, hot embossing of cyclic olefin polymer (COP) can besubstituted for PMMA to form the microfluidic structures of the array.Alternatively, the microfluidic device can be assembled (e.g., prior torunning experiments) by coupling (e.g., uniformly pressing) a substrate120 containing the microstructures against an elastomeric substratewithout permanently adhering to a laminate.

The array 110 is preferably substantially linear with a substantiallyconstant width, but can alternatively be nonlinear and/or have avariable width. The array 110 preferably includes a linear inlet channel140, a linear outlet channel 150 arranged parallel to the inlet channel140, and a set of parallel pores 112 arranged therebetween, as shown inFIG. 1C, normal to the inlet channel 140 and the outlet channel 150, ina manner that fluidly couples the inlet channel 140 and the outletchannel 150 to the set of parallel pores 112. However, the array 110 canalternatively be substantially linear with a diverging or convergingwidth, wherein the inlet channel 140 and the outlet channel 150 arearranged at an angle, and consecutive pores 111 have increasing ordecreasing lengths. The array 110 can alternatively be serpentine,boustrophedonic, curvilinear, or be defined any other suitable geometry.

The pores 111 of the array 110 function to capture and retain cells.Preferably, each pore 111 of the set of pores 112 of the array 110function to capture and retain a single cell of interest, thus enablingprocessing and analysis of an individual cell; however, a pore 111 ofthe set of pores 112 can alternatively be configured to prevent cellcapture, or to capture and retain multiple cells. The pores 111preferably include a chamber 113 configured to receive a cell by achamber inlet 114 and hold a cell, and a pore channel 117 fluidlyconnected to the chamber 113 at a chamber outlet 115. The chamber 113preferably has a length that prevents cell egress due to crossflowwithin the inlet channel 140, and a width or a depth that preventsexcessive cell movement but allows for the cell to move enough such thatthe cell does not block the pore-inlet channel junction. Preferably,each chamber is physically coextensive with an adjacent chamber by abarrier configured to substantially block fluid flow (e.g., in adirection parallel to fluid flow through the pore channel 117, in adirection perpendicular to fluid flow through the inlet channel 140);however, in alternative configurations, a region between two or morechambers 113 can be configured to permit fluid flow therethrough, and/ormay not be physically coextensive with an adjacent pore. The end of thepore channel 117 proximal the chamber outlet 115 preferably has a widththat prevents a captured cell of interest 10 from passing through thepore channel 117 to the outlet channel 150, while permitting one or moresmaller sample components (e.g. lysed cells, cellular components,undesired fluid components, etc.) to flow therethrough. The end of thepore channel 117 proximal the chamber outlet 115 is preferably smallerthan the diameter of a captured cell of interest 10, but can have anyother suitable dimension.

The array 110 preferably includes multiple pores 111. For example, anarray 110 can include 100, 1000, 10,000, 1,000,000, or any suitablenumber of pores 220. The pores 111 are preferably fluidly coupled inparallel within the array 110, wherein the longitudinal axes (i.e., alongitudinal axis of symmetry through the chamber inlet, the chamberoutlet, and the pore channel) of adjacent pores 220 are preferablyparallel and evenly spaced. In some variations of the array withparallel pores 111, however, the pores 220 can be arranged at an angleto adjacent pores 220 within the array 110. In alternative variations,the pores 111 can alternatively be fluidly coupled in any other suitableconfiguration within the array (e.g., one or more of the pores can becoupled in series, such that a pore channel is fluidly coupled to achamber inlet of a downstream pore). The pores 111 of an array 110 arepreferably substantially similar or identical, with chambers 113 ofsubstantially the same dimension and pore channels 117 of substantiallythe same dimension. However, an array 110 can have pores 111 withsubstantially different chamber 113 and pore channel 117 dimensions,with varying chamber 113 lengths, chamber 113 widths, chamber 113depths, pore channel 117 lengths, pore channel 117 widths, pore channel117 depths, number of pore channels 117 per pore 111, number of chambers113 per pore 111, or pores 111 that vary along any other suitableparameter. For example, an array 110 can have multiple pores 111arranged in parallel, wherein consecutive pores iii have decreasing porechannel widths (i.e., an upstream pore has a larger dimension than adownstream pore).

The chamber 113 of a pore 111 functions to retain a cell of interest,while allowing undesired sample components to flow through or around thechamber 113. As such, the chamber 113 is preferably fluidly coupled tothe inlet channel 140 and the pore channel 117, which is fluidly coupledto the outlet channel 150. The chamber 113 of a pore 111 can also enableretention and eventual transfer of intracellular components (e.g.,macromolecules, fragments, nucleic acids, proteins) from a pore channel,for instance, during electrophoresis, after a cell captured within thechamber has been lysed. In one variation, as described in the method 200below, a cell of interest can be captured within a chamber 113,encapsulated in an encapsulation matrix to further prevent cell egress,lysed by diffusion of a lysing reagent across the encapsulation matrix,and genetic content of the lysed cell can amplified with amplificationreagents (e.g., for whole genome amplification), which can enableelectrophoretic separation and analysis. However, the chamber 113 canalternatively be configured to capture a desired particle of interestfrom a sample for any other suitable application.

The chamber 113 preferably has a length and width configured to retainan isolated cell, wherein the chamber 113 is dimensioned to prevent cellegress from the chamber 113 due to inlet channel cross-flow. In onevariation, this is achieved by controlling the width to height ratio ofchamber 113. The width to height ratio of the chamber 222 is preferably1 (e.g., in order to accommodate an approximately spherical cell), butcan alternatively be 1.25, 0.5, or any other suitable ratio. The chamber113 is preferably configured to retain a single cell and to preventmultiple cell retention. In one variation, the chamber 222 isdimensioned such that the height/width of the chamber 222 prevents asecond cell from settling toward the chamber outlet 115 proximal thepore channel 117, and the length of the chamber 222 prevents a singlecell egress from the chamber 222 (e.g. the length is longer than thecell diameter), but encourages egress of a second cell from the chamber222 (e.g. the length is longer than the cell diameter, but shorter thantwo cell diameters). However, the chamber 222 can be configured toretain multiple cells. The chamber 113 preferably has a length, widthand depth each from 5-200 microns, but can alternatively have any othersuitable dimensions. In one variation, the chamber has a length of 30micrometers, a width of 30 micrometers, and a height of 30 micrometers.In another variation, the chamber has a length of 25 micrometers, awidth of 25 micrometers, and a height of 30 micrometers. The chamber 113preferably has a substantially constant cross-section, but canalternatively have a tapering cross-section, preferably that is wider atthe chamber inlet 114 and narrower at the chamber outlet 115. Thevariable cross-section can be the cross-section parallel to the broadface of the substrate 120 and/or the cross-section perpendicular to thelongitudinal axis of the chamber 113. In one variation, as shown in FIG.2A, the chamber 113 has a rectangular cross-section, wherein the porechannel 117 is coupled to the chamber outlet 115, which opposes thechamber inlet 114 coupled to the inlet channel 140. In anothervariation, the chamber 113 has a parabolic cross section, as shown inFIG. 2B and FIG. 2C, wherein the pore channel 117 connects to the apexof the parabolic profile of the chamber 113 at the chamber outlet 115.In another variation, as shown in FIG. 2D, the chamber cross sectionlinearly decreases from the inlet channel 140 to the pore channel 117.In another variation, as shown in FIG. 2E, the chamber cross-sectiondecreases stepwise from the inlet channel 140 to the pore channel 117.In this variation, the chamber 113 defines multiple sub-chambers,wherein the multiple sub-chambers are preferably fluidly connected inseries, wherein a first sub-chamber is fluidly connected to the inletchannel 140 and the last sub-chamber is fluidly connected to the porechannel 117. The first sub-chamber preferably has the largest widthand/or depth, and the last sub-chamber preferably has the smallest widthand/or depth. The transition between the inlet channel 140 and thechamber 113 preferably exhibits a convex angle (e.g. a 90° angle), butcan alternatively be curvilinear as shown in FIG. 2C, or defined by anyother suitable path. The transition between the chamber 113 and the porechannel 117 preferably also exhibits a convex angle (e.g. a 90° angle),but can alternatively be curvilinear or defined by any other suitablepath.

The pore channel 117 of the pore 113 functions to enable retention of acaptured cell of interest 10 and to allow smaller sample components toflow through. The pore channel 117 is preferably fluidly connected tothe chamber outlet 115 and the outlet channel 150. The pore channel 117is preferably substantially straight and linear, but can alternativelybe curvilinear or be defined by any other suitable geometry. The porechannel 117 preferably has a width smaller than the diameter of the cellof interest 10, such that the pore channel 117 prevents passage of acell of interest therethrough. The pore channel 117 preferably has awidth and depth from 1-25 microns and a length from 5-500 microns, butcan have any other suitable width, depth, and/or length. In onevariation, the pore channel 117 has a width of 7-10 micrometers, a depthof 7-10 micrometers, and a length of 5-50 micrometers. The pore channel117 preferably has a substantially constant cross-section, and in aspecific example, the pore channel 117 has a cross section of 8micrometers×10 micrometers. However, the pore channel 117 canalternatively have a tapering or variable cross section. In one suchvariation, the pore channel 117 can be wider proximal the chamber outlet115 and narrow proximal the outlet channel 150. The pore channel 117 ispreferably aligned with its longitudinal axis parallel with thelongitudinal axis of the chamber 113. More preferably, the pore channel117 is coaxial with the chamber 113. However, the pore channel 117 canbe aligned at an angle with the chamber 113 or have any other suitableconfiguration relative to the chamber 113. Each pore 111 preferablyincludes a single pore channel 117, but can alternatively includemultiple pore channels 117, wherein the multiple pore channels 117preferably extend in parallel from the end of the respective chamber 113proximal the outlet channel 150.

1.2 System—Inlet and Outlet Channels

The inlet channel 140, as shown in FIG. 1B, functions to receive avolume of a biological sample and to distribute the biological sample tothe set of pores 112. In variations of the system 100 that allow forelectrophoretic analysis of a set of particles, the inlet channel 140can additionally or alternatively function to receive and facilitatedistribution of a phase-changing matrix (e.g., gel) that allowsencapsulation of captured cells of interest at the pores 111. The inletchannel 140 preferably includes a first end, a second end, and a channelconnecting the first and second ends. The inlet channel 140 ispreferably coupled to a first port 141 at the first end, is fluidlyconnected to the chambers 113 of the array 110 along the inlet channel140 length, and is preferably coupled to a second port 142 at the secondend, as shown in FIGS. 1C and 3. The inlet channel 140 preferablyincludes a first and/or second valve disposed within the first and/orsecond end (e.g., proximal the first port 141, proximal the second port142), wherein the valves can operate between an open and a closed state,in order to facilitate guidance of sample, reagent, and/or encapsulationmatrix flow. In some variations, the first port 141 can facilitatereception of the biological sample and a matrix for encapsulation ofelements captured in the pores 111, and the second port can facilitatedisplacement of the matrix for encapsulation, prior to gelation orsolidification, in order to form a channel that allows for reagentdiffusion across the matrix. In some variations, however, any of thefirst end and the second end can be sealed by the substrate 120 or canbe sealed by a sealant, such as a self-sealing laminate (e.g. made ofrubber, polyethylene, etc.). The body of the inlet channel 140 ispreferably defined by the substrate 120, but can alternatively bepartially defined by the substrate 120, wherein the other portions canbe defined by self-sealing laminate or any other suitable sealant.

The inlet channel 140 is preferably arranged such that a longitudinalaxis of the inlet channel 140 is perpendicular to the longitudinal axesof the chambers 113; however, the inlet channel 140 can alternatively bearranged at an angle relative to the chambers. The chambers 113preferably extend from a single side of the inlet channel 140, but canalternatively extend from multiple sides (e.g. opposing sides) of theinlet channel 140. The inlet channel 140 is preferably substantiallystraight, but can alternatively be curved, bent, or defined by any othersuitable geometry. The inlet channel 140 preferably has a substantiallyconstant rectangular cross-section, but can alternatively have avariable cross section (e.g., a cross-section parallel to the inletchannel longitudinal axis and/or a cross-section perpendicular to theinlet channel longitudinal axis can be constant or variable) that isdefined by any other suitable geometry (e.g., polygonal, curvilinear).In one variation, the inlet channel 140 tapers with distance away fromthe first port 141. The inlet channel 140 preferably has a depth andwidth larger than the diameter of the cell of interest 10, such thatcells of interest can flow freely through the inlet channel 140 withoutundergoing deformation; however, the inlet channel can be dimensionedrelative to a cell of interest in any other suitable manner. The inletchannel 140 preferably a depth and/or width between 5-200 microns, butcan alternatively have any suitable depth and/or width. In onevariation, the inlet channel has a width of 50-100 micrometers, and adepth of 50-100 micrometers, and in a specific example, the inletchannel 140 has a cross sectional dimensions of 100 micrometers by 100micrometers. The inlet channel 140 preferably has a length that canaccommodate all the pores 111 of the array 110; however, in somevariations, the inlet channel 140 can feed a portion of the set of pores112, and not directly be coupled to remaining pores of the set of pores112. In one variation, the inlet channel 140 preferably has a lengthlonger than the combined widths of the chambers 113, such that thechambers 113 are spaced apart from each other (e.g., with uniform ornon-uniform barriers to fluid flow). In another variation, the inletchannel 140 extends to the edge of the substrate 120. However, the array110 can include any suitable configuration of inlet channels 240.

The outlet channel 150, as shown in FIG. 1C, functions to receive andtransmit undesired components of a volume of a biological sample passedthrough the inlet channel 140 and transmitted through the set of pores112. As such, the outlet channel 150 can allow transmission of “waste”fluid from the substrate 120, and/or transmission of biological samplecomponents, omitting the cells of interest, for further processing andanalysis. In variations of the system 100 allowing for electrophoreticanalysis of particles, the outlet channel 150 can additionally oralternatively facilitate transfer of excess encapsulation matrix and/orsieving matrix from the substrate 120, and can additionally oralternatively facilitate transfer and distribution of a sieving matrixfor electrophoresis. The outlet channel 150 preferably includes a firstend, a second end, and a channel connecting the first and second ends.The outlet channel 150 is preferably coupled to a third port 153 at thefirst end, is fluidly connected to the pore channels 117 of the array110 along the outlet channel 150 length, and is preferably coupled to afourth port 154 at the second end of the outlet channel 150, as shown inFIGS. 1C and 3. The outlet channel 150 preferably includes a firstand/or second valve disposed within the first and/or second end (e.g.,proximal the third port 153, proximal the fourth port 154) of the outletchannel 150, wherein the valves can operate between an open and a closedstate, in order to facilitate guidance of sample waste, excess reagent,and/or excess sieving matrix flow. In some variations, the third port153 can facilitate reception of a sieving matrix for electrophoresis,and the fourth port 154 can facilitate transfer of excess reagents,waste, undesired sample components, and/or any other suitable type ofmatter from the substrate 120. In other variations, however, the firstend of the outlet channel 150 and/or the second end of the outletchannel 150 can be sealed by the substrate 120 or can be sealed by asealant, such as a self-sealing laminate (e.g. made of rubber,polyethylene, etc.). Similar to the inlet channel 140, the body of theoutlet channel 150 is preferably defined by the substrate 120, but canalternatively be partially defined by the substrate 120, wherein theother portions can be defined by self-sealing laminate or any othersuitable sealant.

The outlet channel 150 is preferably arranged such that a longitudinalaxis of the outlet channel 150 is perpendicular to the longitudinal axesof the chambers 113; however, the outlet channel 150 can alternativelybe arranged at an angle relative to the chambers 113 of the array 110.Similar to the inlet channel 140, the chambers 113 preferably extendfrom a single side of the outlet channel 150, but can alternativelyextend from multiple sides (e.g. opposing sides) of the outlet channel150. The outlet channel 150 is preferably substantially straight, butcan alternatively be curved or bent, or defined by any other suitablegeometry. The outlet channel 150 preferably has a substantially constantrectangular cross-section, but can alternatively have a variable crosssection (e.g., the cross-section parallel the outlet channellongitudinal axis and/or the cross-section perpendicular the outletchannel longitudinal axis can be constant or variable) that is definedby any other suitable geometry (e.g., polygonal, curvilinear). In onevariation, the outlet channel 150 tapers with distance away from theoutlet third port 153. The outlet channel 150 preferably has a depth andwidth similar to that of the inlet channel 140, but can alternativelyhave a depth and width smaller or larger than that of the inlet channel140. The outlet channel 150 preferably a depth and/or width between5-200 microns, but can alternatively have any suitable depth and/orwidth. In one variation, the outlet channel has a width of 50-100micrometers, and a depth of 50-100 micrometers, and in a specificexample, the outlet channel 150 has cross sectional dimensions of 100micrometers by 100 micrometers. The outlet channel 150 preferably has alength that can accommodate all the pores 220 of the array no. In onevariation, the outlet channel 150 preferably has a length longer thanthe combined widths of the chambers 113, such that the chambers 113 arespaced apart by barriers to fluid flow. In another variation, the outletchannel 150 extends to the edge of the substrate 120.

In some variations, the system 100 can further include at least one ofan inlet manifold configured to couple to the inlet channel 140 (e.g.,at one of the first port 141 and the second port 142) and an outletmanifold configured to couple to the outlet channel 150 (e.g., at one ofthe third port 153 and the fourth port 154). The inlet manifoldfunctions to receive a volume of a biological sample and to distributethe sample to the arrays 200, and the outlet manifold functions tofacilitate transfer of undesired biological sample components and/orexcess matrices for encapsulation/electrophoresis from the substrate120. The inlet manifold and/or the outlet manifold can be that describedin U.S. Pub. No. 2013/0190212, entitled “Cell Capture System and Methodof Use” filed 25 Jul. 2012, which is incorporated herein in its entiretyby this reference; however, the inlet manifold and/or the outletmanifold can alternatively be any other suitable inlet manifold/outletmanifold.

1.3 System—Electrophoresis

As shown in FIGS. 1B and 4, the system 100 can further include a set ofelectrophoresis channels. The set of electrophoresis channels functionto receive a sieving matrix and facilitate electrophoretic separation ofprocessed intracellular content from the cells of interest captured atthe set of pores 112 of the array 110. The set of electrophoresischannels 160 is preferably fluidly coupled to the pore channels 117 ofthe array 110 by the outlet channel 150; however, the set ofelectrophoresis channels 160 can be fluidly coupled to the pore channels117 in any other suitable manner. Preferably, each electrophoresischannel 161 of the set of electrophoresis channels 160 is paired with apore 111 of the set of pores 112, in a one-to-one manner; as such, eachpore channel 117 of the array 110 is preferably aligned with acorresponding electrophoresis channel 161 of the set of electrophoresischannels 160, in order to facilitate electrophoretic separation along alinear path. In specific examples, the system 100 can include 100, 1000,10,000, 1,000,000, or any suitable number of electrophoresis channels161 to match the number of pore channels 117 in the array. However, theset of electrophoresis channels 160 can be configured relative to thepore channels 117 of the array 110 in a manner that is not one-to-one(e.g., contents of multiple pore channels can feed into a singleelectrophoresis channel, an electrophoresis channel can be sufficientlywide to span multiple pore channels, etc.), in a manner wherein theelectrophoresis channels 160 are not aligned with the pore channels 117,along a nonlinear path, and/or in any other suitable manner.

As shown in FIG. 1B, each electrophoresis channel 161 in the set ofelectrophoresis channels 160 preferably includes an electrophoresisinlet 163 proximal the outlet channel 150 and aligned with a porechannel 117, and an electrophoresis outlet 164. The electrophoresisinlets 163 can be partially separated from the outlet channel 150 by aporous membrane 169, as shown in FIG. 1B, configured to block a majorityof fluid flow from the outlet channel (e.g., such that a majority of thefluid flows out of the fourth port 154 without entering theelectrophoresis inlets 163), but that still allows a conductiveinterface to form between an encapsulation matrix and a sieving matrixdelivered into the substrate 120. However, the electrophoresis inlets163 and the outlet channel 150 can be separated in any other suitablemanner, and/or not separated by a membrane. The electrophoresis inlet163 functions to receive processed intracellular components that areelectrokinetically driven by an electric field, and the electrophoresisoutlet 164 functions to facilitate distribution of a sieving matrix forelectrophoresis, such that intracellular macromolecules and fragments(e.g., proteins, nucleic acids) can be separated along an entire lengthof an electrophoresis channel 161. The length of the electrophoresischannel 161 thus preferably defines a length that allows for separationof macromolecules and fragments with proper resolution (e.g., clearseparation of bands characterizing specific macromolecules andfragments), and in one variation, is minimized to contribute tocompactness of the system 100. However, in other variations, the lengthof an electrophoresis channel 161 can be any other suitable length(e.g., not minimized), for example, in applications wherein compactnessis less of a concern. As such, a region between each electrophoresisinlet 163 and electrophoresis outlet 164 functions to provide a pathwayalong which intracellular macromolecules and fragments can be separatedand analyzed with suitable band resolution. Preferably, thecross-section of an electrophoresis channel 161 defines a rectangulargeometry with a low aspect ratio; however, an electrophoresis channel161 can alternatively have any other suitable cross-sectional geometrydefining any other suitable aspect ratio.

Each electrophoresis channel 161 is preferably defined within thesubstrate 120 using techniques identical to that of forming at least oneof the array 110, the inlet channel 140, and the outlet channel 150,such that processing of the set of electrophoresis channels 160 can beperformed simultaneously with at least one of the array 110, the inletchannel 140, and the outlet channel 150. However, the set ofelectrophoresis channels 160 can be performed in any other suitablemanner. In one specific example, the electrophoresis channels 161 areprocessed simultaneously with the array 110, the inlet channel 140, andthe outlet channel, using a three mask photolithographic process anddeep reactive ion etching (DRIE) process to etch the set ofelectrophoresis channels 160 into a silicon or glass substrate as amold. In the specific example, the etched elements are then transferredto 1 millimeter thick polymethylmethacrylate (PMMA) sheets as asubstrate 120 using a hot embossing process, which is then laminatedwith a polymethylmethacrylate (PMMA) laminate to define the set ofelectrophoresis channels 160. In the specific example, laminationincludes utilizing an appropriate roller speed, temperature, pressure,and tension of the laminate to ensure a low level of ingress of laminatematerial into microfluidic structures. The chamber 113 preferably has awidth and depth each from 5-200 microns, but can alternatively have anyother suitable dimensions. In a specific example, each electrophoresischannel 161 has a length of approximately 15 millimeters, a width of 30micrometers, and a depth of 8 micrometers. As such, the specific exampleof the set of electrophoresis channels 160 provides channels forelectrophoretic separation with a length for suitable band resolution,and a cross-section with a low aspect ratio that facilitatesvisualization of bands.

As shown in FIG. 4, each electrophoresis outlet 164 of the set ofelectrophoresis channels 160 is preferably fluidly coupled to anelectrophoresis outlet channel 167, which functions to facilitatedistribution of a sieving matrix throughout the set of electrophoresischannels 160 for electrophoresis, and facilitate distribution ofreagents (e.g., separation buffer) throughout the system 100 forprocessing of the set of cells. The electrophoresis outlet channel 167preferably includes a first end, a second end, and a channel connectingthe first and second ends. The electrophoresis outlet channel 167 ispreferably coupled to a fifth port 165 at the first end, is fluidlyconnected to the electrophoresis outlets 164 of the set ofelectrophoresis channels 160 along the electrophoresis outlet channel167 length, and is preferably coupled to a sixth port 166 at the secondend of the electrophoresis outlet channel 167, as shown in FIG. 1B. Theelectrophoresis outlet channel 167 preferably includes a first and/orsecond valve disposed within the first and/or second end (e.g., proximalthe fifth port 165, proximal the sixth port 166) of the electrophoresisoutlet channel 167, wherein the valves can operate between an open and aclosed state, in order to facilitate guidance of excess reagent and/orexcess sieving matrix flow. In some variations, the fifth port 165 canfacilitate reception of a buffer (e.g., a separation buffer) that istransferred throughout the sieving matrix for electrophoresis, and thesixth port 166 can facilitate transfer of excess reagents, excesssieving matrix and/or any other suitable type of matter from thesubstrate 120. In relation to the inlet channel 140, the outlet channel150, and the electrophoresis outlet channel 167, any one or more of thefirst port 141, the second port 142, the third port 153, the fourth port154, the fifth port 165, and the sixth port 166 can facilitate receptionof a buffer (e.g., a separation buffer) that is transferred throughoutthe sieving matrix for electrophoresis. Furthermore, the system 100 caninclude any other suitable number of ports (e.g., coupled to the inletchannel, coupled to the outlet channel, coupled to the electrophoresisoutlet channel, defined within any other suitable location of thesubstrate) configured to facilitate processing of the set of cells. Inother variations, however, the first end of the outlet channel 150and/or the second end of the electrophoresis outlet channel 167 can besealed by the substrate 120 or can be sealed by a sealant, such as aself-sealing laminate (e.g. made of rubber, polyethylene, etc.). Similarto the inlet channel 140 and the outlet channel 150, the body of theelectrophoresis outlet channel 167 is preferably defined by thesubstrate 120, but can alternatively be partially defined by thesubstrate 120, wherein the other portions can be defined by self-sealinglaminate or any other suitable sealant.

Also shown in FIG. 1B, the system 100 can further include a set ofelectrodes 170. The set of electrodes 170 function to provide anelectric field across the substrate 120 in a manner that facilitateselectrokinetic movement of processed intracellular content from thecells of interest captured at the set of pores 112 of the array 110,through the set of electrophoresis channels 160. Preferably the set ofelectrodes includes a first electrode 171 configured to provide apositive voltage and a second electrode 172 configured to provide anegative voltage, such that an electric field is created between thefirst electrode 171 and the second electrode 172, as shown in FIGS. 1Band 4. Preferably, the first electrode 171 is configured proximal to alocation upstream of the set of pores, and the second electrode 172 isconfigured proximal to a location downstream of the set ofelectrophoresis channels. In one variation, the first electrode 171 iscoupled to the substrate 120 proximal the inlet channel 140, and thesecond electrode 172 is coupled to the substrate 120 proximal theelectrophoresis outlet channel 167 and the electrophoresis outlets 164of the set of electrophoresis channels 160, such that intracellularmacromolecules and fragments can be electrokinetically driven from thechambers 113 of the array 110, through the pore channels 117, andthrough the set of electrophoresis channels 160 in an electrophoresisinlet-to-electrophoresis outlet direction. In another variation, thefirst electrode 171 can be coupled to the substrate 120 proximal thechambers 113 of the array, and in yet another variation, the firstelectrode 171 and the second electrode 172 can be coupled to thesubstrate at opposing peripheral regions of the substrate 120; however,in other variations, the set of electrodes 170 can be configured in anyother suitable manner relative to the substrate.

The set of electrodes 170 preferably includes electrically conductiveelements that can be coupled to a source configured to generatespecified voltages. In variations, the electrically conductive elementscan include any one or more of: composite materials, alloys, purematerials, and any other suitable electrically conductive material.Furthermore, the electrically conductive elements are preferably wires;however, the electrically conductive elements can alternatively bedefined by any other suitable form factor (e.g., particulate, sheet,etc.). The set of electrodes 170 can be coupled to the substrate usingany suitable process, and in variations, can be coupled using any one ormore of: lamination, a thermal bonding method, and adhesives to providerobust coupling. In a specific example, the set of electrodes 170includes gold-coated copper wires that are 0.1 millimeters in diameter,which are laminated between the PMMA substrate 120 and the PMMA laminateproximal the inlet channel 140 and the electrophoresis outlet channel167, with electrically conductive epoxy that provides electricalcontacts for microelectrophoresis. However, the set of electrodes 170can include any other suitable number of electrodes, and can beconfigured relative to the system 100 in any other suitable manner.

1.4 System—Additional Elements

As shown in FIGS. 5A and 5B, the system 100 can additionally include anencapsulation module 500 that functions to encapsulate cells and/orother captured particles (e.g., reagent particles) within individualpores 111. In one variation, the encapsulation module 500 can implementany one or more of the first port 141, the second port 142, the thirdport 153, the fourth port 154, the fifth port 165, and the sixth port166, in order to isolate particles at the pores 111 of the array 110. Inone variation, an encapsulation matrix 501 can be flowed through thefirst port 141, into the inlet channel 140, through the pores 111, andout of the outlet channel 150 to the fourth port 154, forming a firstencapsulation layer 502 between the set of pores 112 and the inletchannel 140, and a second encapsulation layer 503 between the porechannels 117 of the array 110 and the outlet channel 150. Theencapsulation layers are preferably 10 to 20 micrometers thick, but canalternatively be thicker or thinner. During encapsulation matrixintroduction, buffer is preferably simultaneously flowed through theinlet channel 140 and outlet channel 150, preferably in the samedirection as encapsulation matrix flow, wherein the buffer flow ratepreferably controls the thickness of the encapsulation matrix layers502, 503. Buffer flow is preferably established in the portions of theinlet channel 140 and outlet channel 150 distal from the pores 220. Thebuffer flow rate is preferably maintained at laminar flow, but canalternatively have any other suitable flow rate. However, any othersuitable mechanism that can establish a first and second encapsulationlayer can be used.

The encapsulation matrix 501 preferably isolates a pore 117 within anarray 110. The encapsulation matrix 501 preferably has a flow state anda set state, wherein a photochemical reaction, phase transition,thermochemical reaction, polymerization reaction or any other suitablereaction switches the encapsulation matrix from the flow state to theset state. In the flow state, the encapsulation matrix 501 is preferablysubstantially viscous, such that the encapsulation matrix 501 does notflow into the pores 111 during introduction into the system 100. In theset state, the encapsulation matrix 501 is preferably a solid or gelthat prevents particle egress from the pores 111 (e.g., egress of cellsand large nucleic acid molecules from the pores), and is preferablyporous or selectively permeable to permit small molecule, buffer, andreagent penetration therethrough. In one variation, the encapsulationmatrix 501 is a microporous agarose gel, and in another variation, theencapsulation matrix is a photopolymerizable hydrogel, such as PEG orpolyacrylamide with photoinitiator; however, the encapsulation matrixcan alternatively be any suitable material with any other suitablepolymerization agent. In some variations, select portions of theencapsulation matrix 501 can be reacted to seal specific pores 111. Forexample, as shown in FIG. 5C, a unique photomask 504 can be created thatallows collimated irradiation of encapsulation matrix segments blockingpores 111 containing the cells of interest, while leaving pores void ofcells of interest not encapsulated. The photomask 504 can be created byhigh resolution printing of UV-blocking black ink on a transparencysheet or by use of standard photolithography on photoresist coated glassmasks. The selective UV exposure of select regions of the microfluidicchip can also be accomplished by moving a UV laser or a collimated andconcentrated UV spot to the select locations using an x-y stage.Undesired sample components 20 and unreacted encapsulation matrix 501can then be removed from the system 100 by ingressing fluid through theoutlet channel 150 (e.g. backflowing) and/or the inlet channel 140.Alternatively, the photomask 504 can allow irradiation of encapsulationmatrix segments blocking pores 111 containing undesired samplecomponents 20, wherein desired cells 10 are retrieved from the system.However, any suitable portion of the encapsulation matrix 501 can bereacted. In one alternative variation, a molten encapsulant can be flowninto desired portions (e.g., a portion or all the microfluidic network),and the molten encapsulant can be transitioned to a set-stage. In thealternative variation, an irradiation device (e.g., an infrared laser)can then be used to irradiate desired regions of the microfluidicnetwork to melt desired sections and create a desired flow path.

In some variations, as shown in FIGS. 5A and 6, the encapsulation module500 can further facilitate distribution of a sieving matrix 511throughout the system 100 (e.g., the set of electrophoresis channels160), in order to provide a continuous matrix that allows for separationand analysis of intracellular components by electrophoresis. The sievingmatrix 511 can be identical in composition to the encapsulation matrix501, or can be non-identical in composition to the encapsulation matrix501. Preferably, the sieving matrix 511 is configured to provide acontinuous interface with the encapsulation layers formed by theencapsulation matrix 501; however, the sieving matrix 511 canalternatively be configured in any other suitable manner. In oneexample, the encapsulation module 500 can utilize the third port 153 andthe sixth port 166, in distributing a sieving matrix across the set ofelectrophoresis channels 160; however, other variations can use anyother suitable port for transferring sieving matrix into the system 100.

The system 100 can additionally include optical elements 180 thatfunction to facilitate imaging. The optical elements 180 function toadjust incoming light, preferably to facilitate better imaging. Theoptical elements 180 can function to bend, reflect, collimate, focus,reject, or otherwise adjust the incoming light. The optical elements 180are preferably fabricated within the same process as the system 100manufacture, but can alternatively be included after system 100manufacture. The optical elements 180 are preferably defined within thesubstrate 120, but can alternatively be defined by any other suitablecomponent of the system 100. Optical elements 180 can include lightreflectors disposed within the substrate thickness adjacent the array(s)110 (as shown in FIG. 7A), defined on a broad face of the substrate 120opposite that defining the array 110 (as shown in FIG. 7B), ormicrolenses defined on a broad face of the substrate proximal thatdefining the array 110 (as shown in FIG. 7C), light collimators, lightpolarizers, interference filters, 90° illumination, elements thatminimize excitation rays from going into path of collected fluorescenceemission light, diffraction filters, light diffusers, or any othersuitable optical element. In one such variation, the substrate canfurther include a reflector, separated from the inlet channel by an airgap and configured to reflect incident light at a 90 degree anglelongitudinally into each pore of the set of parallel pores, as shown inFIG. 7D. Alternatively, the optical elements 180 can be defined by animaging stage or by any external component.

The system 100 can additionally include pore affinity mechanisms thatfunction to attract a cell of interest 10 towards a pore 111. Poreaffinity mechanisms can include electric field traps, features withinthe inlet channel 140 that direct flow into a pore 111, negativepressure application to the outlet channel 150, or any other suitablepore affinity mechanism.

In some variations, the system 100 can further be configured tofacilitate selective cell removal from known, addressable locations.While an individual cell from a single pore 111 is preferablyselectively removed, the system can facilitate simultaneous removal ofmultiple cells from a single array 110. The cell is preferably removedby applying a removal force to a cell captured within a chamber 113. Theremoval force is preferably applied by pumping fluid through the porechannel 117 into the chamber 113, but can alternatively be applied byaspirating the contents out of the chamber 113. In one variation, thepump pressure provided by a pump mechanism at an outlet of the system100 is less than 10,000 Pa, in order to prevent damage to a cell beingretrieved. In one specific variation, the provided pump pressure is6,000 Pa. However, any other suitable pump or aspiration pressure can beused. In some variations, cell removal can be achieved by utilizing acell removal tool 600. The cell removal tool 600 of the system 100functions to selectively remove one or more isolated cells from anaddressable location within the system 100. The cell removal tool 600 ispreferably configured to remove a cell from a single chamber 113, butcan alternatively be configured to simultaneously remove multiple cellsfrom multiple chambers 113. In some variations, the cell removal toolcan additionally or alternatively be configured to selectively deliverspecific reagents (e.g., cell lysis reagents, nucleic acid bindingreagents/particles, biomarker binding or detection reagents, etc.) toselect cells and/or can be used to selectively remove cellularcomponents, such as cell lyate, nucleic acid from select cells. In onevariation, the cell removal tool 600 is configured to remove one or morecells from the system 100 in a direction substantially parallel to thebroad face of the substrate 120. As shown in FIGS. 8A and 8B, the cellremoval tool 600 preferably includes a cannula 680 defining a lumen andan aperture 684. The cannula 680 preferably terminates in a sealedpuncture tip 682 at a first end, and is preferably fluidly connected toa cell collection volume at a second end. The aperture 684 is preferablya hole that extends through the cannula 680 wall, wherein the holepreferably has a width substantially equivalent to or larger than thewidth of a pore chamber 222, but small enough such that the aperture 684does not span two pore chambers 113. The cannula 680 preferably includesone aperture 684, but can alternatively include multiple apertures 684,wherein the multiple apertures 684 can be aligned in a line parallel tothe longitudinal axis of the cannula 680, or can be distributed aboutthe surface of the cannula 680 (e.g. spiral about the longitudinal axisof the cannula 680). The aperture 684 preferably extends through alongitudinal cannula 680 wall, but can alternatively extend through aportion of the puncture tip 682. In one example, the aperture 684extends through a portion of the longitudinal cannula wall proximal thepuncture tip 682. In another example, the aperture 684 extends through aportion of the longitudinal cannula wall a predetermined distance fromthe puncture tip 682, wherein the distance can be configured such thatthe cannula wall blocks one or more of the adjacent pores 220. Inanother example, the aperture 684 can extend through the puncture tip682 such that the longitudinal axis of the aperture 684 extends inparallel or coaxially with the longitudinal axis of the cannula 680. Thetransition between the aperture 684 and the cannula 680 exterior and/orinterior is preferably convex and curved to prevent cell damage, but canalternatively be concave, angled, be at right angles, or have anysuitable configuration. The cannula 680 preferably has a circular crosssection, but can alternatively have a rectangular or square crosssection, ovular cross section, or any other suitable cross section. Thecannula 680 is preferably rigid, but can alternatively be flexible orinclude flexible portions. In one alternative, the cannula 680 isflexible and includes a rigid puncture device 686, wherein the rigidpuncture device 686 is slidably coupled over the cannula 680. The rigidpuncture device 686 forms and retains an entryway into the inlet channel140, and the cannula 680 can be advanced therethrough. However, thecannula 680 can have any other suitable configuration. The cannula 680can additionally include a perforator slidably coupled within the lumen,wherein the perforator can extend through the aperture 684 to perforateany intermediary layers between the cannula 680 and the pore 111 (e.g.an encapsulation layer). The perforator position post perforation can beretained to facilitate cell removal therethrough, or the perforator canbe retracted prior to cell removal.

In one variation of cell retrieval tool operation, the cannulapreferably traverses through the inlet channel 140 of the array 110(e.g., through one of the first port 141 and the second port 142,through a side adjacent to or opposing a broad surface of the substrate120), until the aperture is aligned with the pore 111 containing thecell of interest 10. The inlet channel can thus function as a guide toguide the cell removal tool to a pore, and in variations wherein thesystem 100 includes arrays coupled in series, inlet channels fordifferent arrays can be configured to guide the cell removal tool forextraction of a captured cell, as shown in FIG. 8C. Fluid can then beingressed through an outlet manifold coupled to an outlet channel 150 ofan array 110, wherein the pressure of the ingressed fluid pushes thecell of interest 10 out of the pore chamber 113, through the aperture684, and into the cannula. Subsequent fluid ingress through the inletchannel 140 can recapture any cells that were backflowed out of theirrespective pores 111. The cannula can additionally or alternativelyinclude a low-pressure generation mechanism fluidly coupled to the lumenthat aspirates the cell out of the pore 111. Alternatively oradditionally, the cannula can facilitate cell ingress through capillaryaction. The cell preferably travels through the lumen and is storedwithin the cell collection volume.

In this variation of cell retrieval tool operation, the cannula ispreferably inserted into the inlet channel 140 through the side of thesubstrate 120, as shown in FIG. 8B, wherein the inlet channel 140preferably partially defined by a self-sealing portion (e.g., aself-sealing wall) that provides a hermetic seal about the cell removaltool upon penetration of the self-sealing portion. One or more inletchannels 140 coupled to an array 110 can further be substantiallyaligned with a guide 650 that facilitates guidance of the cell removaltool 600 into a respective inlet channel 140 for retrieval of a capturedcell 10, wherein the guide 650 is separated from a respective inletchannel 140 by the self-sealing portion. The cannula is preferablyextended through this self-sealing portion in order to access a capturedcell of interest. Alternatively, the cannula can be inserted into theinlet channel 140 through a top layer of the substrate 120, wherein thecannula can be flexible to accommodate the angle of entry, or the toplayer can be elastic to accommodate the angle of entry. However, anyother suitable method of introducing the cannula into the inlet channel140 can be used, and introduction can be facilitated by use of aprecision stage (e.g., a precision x-y stage) supporting the substrate,wherein positions of the precision stage can be manually and/orautomatically adjusted.

In another variation of cell retrieval tool operation, the cannulaincludes an aperture through the puncture tip. The cannula is advancedthrough the inlet channel 140, successively blocking each successivepore chamber 113 until only the desired subset of pores 111 are leftuncovered. Fluid can then be provided through the outlet channel 150directly fluidly connected with the uncovered pores 111 tosimultaneously release the cells from the uncovered pores 111, whereinthe fluid preferably entrains the cells and moves the cells into thecannula. The cannula can additionally or alternatively be fluidlyconnected to a low-pressure generator to aspirate the cells into thecell collection volume.

Cell removal from the system 100 is preferably automated, but canalternatively be semi-automated or manual. Cell identification caninclude automatic fixing, permeabilization, staining, imaging, andidentification of the cells through image analysis (e.g. through visualprocessing with a processor, by using a light detector, etc.). Cellremoval can include advancement of a cell removal tool 600 to the pore111 containing the cell of interest 10. Cell removal can additionallyinclude cell removal method selection and/or cell removal toolselection. In another variation, cell identification can semi-automated,and cell retrieval can be automated. For example, cell staining andimaging can be done automatically, wherein identification and selectionof the cells of interest can be done manually. In another variation, allsteps can be performed manually. However, any combination of automatedor manual steps can be used. Furthermore, in other variations, the cellremoval tool 600 and/or cell removal operations can include any othersuitable tool or operation, such as those described in U.S. Pub. No.2013/0190212, entitled “Cell Capture System and Method of Use” filed 25Jul. 2012, which is incorporated herein in its entirety by thisreference.

1.5 System—Examples

In an example, as shown in FIG. 1B, the system 100 includes an array 110including a plurality of 1000 substantially identical pores 111, eachconnected to an inlet channel 140 at the chamber inlet 114 and an outletchannel 150 at the pore channel 117. In the example, each pore 111 ispaired with and substantially co-aligned with an electrophoresis channel161, such that there are 1000 electrophoresis channels in parallel,fluidly coupled to the outlet channel 150. Each of the electrophoresischannels defines an electrophoresis inlet 163 and an electrophoresisoutlet 164, has a substantially constant rectangular cross-section alongits length, and is substantially linear (e.g., without any curvedportions). Furthermore, each electrophoresis outlet 164 is fluidlycoupled to an electrophoresis outlet channel 167 to facilitatedistribution of a sieving matrix throughout the system 100. In theexample, the outlet channel 150 includes a first port 141 and a secondport 142, the outlet channel 150 includes a third port 153 and a fourthport 154, and the electrophoresis outlet channel 167 includes a fifthport 165 and a sixth port 166, wherein each of the ports 141, 142, 153,154, 165, 166 is in communication with a valve, in order to enabledirected transmission of biological samples, fluids, reagents, andmatrices throughout the system 100. The array 110, inlet channel 140,outlet channel 150, electrophoresis channels 160, and electrophoresisoutlet channel 167 are preferably recesses defined on one broad face ofa PMMA substrate 120, formed by hot-embossing a PMMA sheet on an etchedsilicon mold and are preferably cooperatively defined by a top layer ofPMMA laminate that fluidly seals microfluidic structures. The set ofelectrodes 170 in the example includes gold-coated copper wires that are0.1 millimeters in diameter, which are laminated between the PMMAsubstrate 120 and the PMMA laminate proximal the inlet channel 140 andthe electrophoresis outlet channel 167, with electrically conductiveepoxy that provides electrical contacts for microelectrophoresis. In theexample, the inlet channel 140 and the outlet channel 150 each have adepth and width of 100 micrometers, the chambers 113 of the pores 111each have a depth and width of 30 micrometers, the pore channels 117each have a depth and a width of 8 micrometers, and the electrophoresischannels 161 each have a depth of 8 micrometers, a width of 30micrometers, and a length of 15 millimeters.

In another example, as shown in FIG. 9, the system 100 includes aplurality of substantially identical arrays 110 arranged in parallel; aninlet port 141 coupled to an inlet channel 140, and an outlet port 154coupled to an outlet channel 150. The plurality of arrays includes aplurality of 100,000 substantially identical pores 111 connected to arespective inlet channel 140 at the chamber 113 and a respective outletchannel 150 at the pore channel 117. Each inlet channel 140 issubstantially aligned with a guide 650 that facilitates guidance of acell removal tool 600 into a respective inlet channel 140 for retrievalof a captured cell 10, wherein the guide 650 is separated from arespective inlet channel 140 by a self-sealing barrier configured toform a hermetic seal about the cell removal tool 600 upon penetration.The system 100 in this example allows up to 5 mL of blood to be receivedat a substantially low pressure (e.g., <10 kPa) in less than 10 minutes.The arrays 110, inlet channels 140, and outlet channels 150 arepreferably recesses defined on one broad face of a substrate 120, andare preferably cooperatively defined by a top layer (e.g., PMMAlaminate) that fluidly seals the arrays 110, inlet channels 140, andoutlet channels 150 from the system 100 exterior. The inlets channelsand outlet channels are preferably accessible by holes defined throughthe thickness of the substrate 120, and preferably originate from thesubstrate broad face opposing the face defining the arrays 110, inletchannels 140, and outlet channels 150. Additionally or alternatively,the holes can be configured to extend through the substrate 120 from thesubstrate sides and/or in any other suitable manner.

In other embodiments, variations, and examples, the system 100 canfurther include any other suitable elements that facilitate cellprocessing and analysis. Additionally, as a person skilled in the fieldof cell sorting will recognize from the previous detailed descriptionand from the figures and claims, modifications and changes can be madeto the embodiments, variations, examples, and specific applications ofthe system 100 described above without departing from the scope of thesystem 100.

2. Method

As shown in FIGS. 10A and 10B, a method 200 for capturing and analyzinga set of cells comprises: capturing the set of cells S210 at a set ofpores of a substrate, each pore including a chamber configured to hold asingle cell of the set of cells; transmitting a set of reagent particlesto the set of pores S220, wherein the set of reagent particles isconfigured to facilitate whole genome amplification and polymerase chainreaction (PCR) of each cell in the set of cells; encapsulating the setof cells and the set of reagent particles within an encapsulation matrixat the set of pores S230; delivering a lysing reagent across theencapsulation matrix, thereby lysing the set of cells S240; andamplifying nucleic acid content of the set of cells at the set of pores,thereby facilitating analysis of the set of cells S250. In somevariations, the method 200 can further include transmitting a sievingmatrix to a set of electrophoresis channels fluidly coupled to the setof pores S260; and transmitting an electric field across the substrateS270, thereby enabling electrophoretic analysis of the set of cells.

The method 200 functions to enable isolation, capture, and retention ofcells, more preferably single cells, at known, addressable locations,and further to facilitate performance of multiple single-cell assaysthat can be performed on individual cells (e.g., rare cells in abiological sample). The method 200 is preferably implemented at least inpart using the system 100 described in Section 1 above; however themethod 200 can additionally or alternatively be implemented using anyother suitable system 100 for cell capture and analysis. In someembodiments, the method 200 can be used to capture circulating tumorcells (CTCs) and subpopulations of CTCs, such as circulating stem cells(CSCs), but can additionally or alternatively be used to capture anyother suitable cell of possible interest for processing and analysis.

Block S210 recites: capturing the set of cells at a set of pores of asubstrate, each pore including a chamber configured to hold a singlecell of the set of cells, which functions to segregate cells of interestwithin chambers configured to retain a single cell, in order tofacilitate analyses of the set of cells in a single-cell format. BlockS210 is preferably implemented at a set of pores of an embodiment of thearray of the system 200 described in Section 1.1 above. The set of cellsare preferably carried in a volume of a biological sample, and in somevariations, can include a volume of blood or any other suitable digestedtissue. The set of cells are thus cells of interest (e.g., circulatingtumor cells, stem cells, etc.) that are carried in the biologicalsample, but in some variations, can include cells or other particlesthat are spiked into the biological sample (e.g., for researchapplications).

In a specific example, Block S210 includes receiving a biological sample(e.g., a volume of blood collected by venipuncture from donors andstored in EDTA-treated containers, a volume of saline/bovine serumalbumin/EDTA buffer), wherein the biological sample is spiked with anumber (e.g., 1, 5, 50, 100, 200, etc.) breast cancer cell line MCF 7cells. In the specific example, the MCF 7 cells are maintained inEagle's Minimum Essential Media (EMEM) supplemented with 10% fetalbovine serum (FBS) and 100 units per milliliter ofPenicillin-Streptomycin, and grown at 37 C in a humidified incubator(e.g., 95% humidity) in a 5% carbon dioxide environment prior toharvesting and spiking into the biological sample. The biological samplewith the cells of interest is then mixed with fixative (e.g., an equalvolume of 1% paraformaldehyde, equal volume of 2% formalin) and received(e.g., by way of a pump providing less than 1 psi of pumping pressure)at a first port of an inlet channel coupled to the set of pores, andtransmitted through the set of pores to capture the set of cells.Undesired biological sample components are passed through a set of porechannels coupled to the pores, to an outlet channel coupled to a fourthport for waste removal. In the specific example, a pore chamber depth of8 micrometers and a pumping pressure less than 1 psi allows the cancercells of interest (i.e., MCF 7 cells that are 15-30 micrometers indiameter) to be retained at the set of pores, while red blood cells andwhite blood cells pass through and are not captured. In variations ofthe specific example, a priming buffer can be received into the inletchannel and the set of pores prior to reception of the biologicalsample, wherein the priming buffer prevents trapping of air bubbles,which can obstruct sample processing. However, in other variations,Block S210 can be implemented using any other suitable system configuredto capture and isolate cells of interest in a single cell format.

Block S210 preferably includes capturing the set of cells without theuse of affinity molecules configured to bind to a cell of the set ofcells, such that captured cells undergo minimal manipulation and can beretrieved for further processing; however, capturing the set of cells inBlock S210 can alternatively include implementation of any suitableaffinity mechanism, and in some variations, can include any one or moreof: electric field traps, microfluidic features that direct sample fluidflow into a pore, negative pressure application to the outlet channel150, affinity molecules, chemotaxic gradients that attract cells in adesired direction, magnetic tagging and manipulation of tagged particlesby a magnetic field, and any other suitable affinity mechanism. In BlockS210, the set of cells preferably includes CTCs, such that Block S210includes capturing substantially all (e.g., over 85%) CTCs present in abiological sample at the set of pores. However, Block S210 canadditionally include capture of heterogeneous populations of cells, withany suitable efficiency, at a set of pores. Furthermore, some variationsof Block S210 can include capture of multiple cells in a single pore,such that capture is not single-format.

In some variations, as shown in FIG. 10A, Block S210 can further includecapturing a subpopulation of the set of cells at a subset of the set ofpores S212, such that populations and subpopulations of a cell-type ofinterest can be captured in a single-cell format for analysis. In suchvariation, Block S210 can include capturing a set of CTCs at the set ofpores 111 single-cell format, and Block S212 can include capturing asubpopulation of self-renewing cancer stem cells (CSCs), which areassociated with treatment resistance and higher metastatic potential. InBlock S212, the subpopulation of the set of cells is preferably capturedsimultaneously with the set of cells using a set of identical poresbased upon size and deformability properties, with identification andsingle-cell analyses performed in subsequent steps. However, Block S212can be performed non-simultaneously with Block 210, can be performedusing non-identical pores (e.g., a set of pores including poresconfigured to capture CTCs and pores configured to capture CSCs), and/orcan be performed in any other suitable manner.

Block S220 recites: transmitting a set of reagent particles to the setof pores, which functions to deliver activatable reagents to thecaptured cells of interest, prior to encapsulation in Block S230 and/oranalysis to discriminate between captured cells of interest andcontaminants. In variations, the reagent particles can includemicrospheres (magnetic or non-magnetic) containing affinity molecules tobind nucleic acids (e.g., total nucleic acid, DNA, RNA) or nucleic acidcontaining specific oligonucleotide sequences, antibodies, orpolypeptides. In one example, and similar to reception of the set ofcells, the set of reagent particles are received at a first port of aninlet channel coupled to the set of pores, and captured at pores of theset of pores containing a captured cell of the set of cells. In theexample, excess reagent particles are passed through a set of porechannels coupled to the pores, to an outlet channel coupled to a fourthport for waste removal. The set of reagent particles are preferablysized such that the reagent particles are caught between a cell capturedin a pore, and a wall of the pore, but are unable to escape because ofthe presence of the cell abutting a pore channel coupled to the pore;however, the reagent particles can be characterized by any othersuitable property (e.g., adhesive behavior, viscosity, morphology, etc.)that enables delivery and capture of the set of reagent particles atpores containing a captured cell of interest. In one alternativevariation, however, a pore can be configured to uniformly capturereagent particles and cells of interest in any suitable order, forinstance, due to geometric configurations of the pore (e.g., the porecomprises a first compartment that is complementary to a reagentparticle and a second compartment that is complementary to a cell ofinterest, wherein the first compartment and the second compartment arein fluid communication).

In some variations, as shown in FIG. 11, the set of reagent particles isconfigured to facilitate whole genome amplification (WGA) and allelespecific polymerase chain reaction (AS-PCR) or target specific PCR ofeach cell in the set of cells, however, the set of reagent particles canalternatively be configured to facilitate only one of WGA and AS-PCR.Additionally or alternatively, the set of reagent particles can beconfigured to facilitate any other type of genetic amplification (e.g.,for any other type of PCR, for multiple annealing and looping basedamplification cycles, for loop-mediated amplification, fortranscription-mediated amplification, for nucleic acid sequence basedamplification, etc.) in order to amplify content. In variations whereinreagent particles for multiple types of amplification (or otherprocessing) are co-received and transmitted, the set of reagentparticles can include particles of different properties (e.g., meltingtemperatures, etc.) in order to facilitate sequential processing of theset of cells according to the different types of amplification (or otherprocessing). In a specific example, the reagent particles for WGAinclude particles 6 micrometers in diameter composed of low-meltingagarose (e.g., melting point of 65 C) coated with random hexamer primersrequired for WGA. In a specific example, the reagent particles forAS-PCR include particles 6 micrometers in diameter composed ofpolystyrene and processed with conjugated forward primers for AS-PCR.

In some variations, Block S220 can additionally or alternatively includereceiving reagents at the set of pores, wherein the reagents areconfigured to facilitate identification of a subpopulation of the set ofcells captured at a subset of the set of pores (e.g., captured, as inBlock S212). Block S220 can thus include transmitting a reagent volumeto the set of pores and/or can include receiving and transmittingreagents in any other suitable manner. In one variation, the regents caninclude an antibody cocktail configured to facilitate distinguishing ofa subpopulation of cells (e.g., CSCs) from the set of cells (e.g. CTCs),wherein incubation with antibody cocktail can enable identification ofthe subpopulation of cells by fluorescent detection. In examples of thisvariation implemented at an embodiment of the system 100 describedabove, the reagents can be received into the inlet channel (e.g., at thefirst port) coupled to the set of pores, and delivered to captured cellsat the set of pores. In a specific example of this variation, theantibody cocktail can include CD24 and CD44 antibodies, whereinexpression of a CD44⁺/CD24⁻ phenotype facilitates identification of CSCsfrom a set of CTCs. The antibody cocktail in the specific example canfurther include CAM 5.1 (CK8/18) antibodies, which can help distinguishcancer cells (e.g., CTCs, CSCs) of the set of cells from contaminatingcells (e.g., leukocytes). In the specific example, the antibody cocktailis delivered into the first port of an inlet channel fluidly coupled tothe set of pores containing captured cells, with Hoechst nuclear stainas a counter stain. The antibody cocktail is then incubated with the setof cells and the subpopulation of cells, after which fluorescentdetection is used to facilitate retrieval and/or downstream analyses ofthe subpopulation of CSCs. In variations of the specific example,antigen retrieval and/or alternative fixation processes can facilitateprocessing and detection of CSCs of the set of CTCs. In one suchvariation, alternative fixatives (e.g., alternatives to formalin) caninclude −20 C methanol, acetone, and 1:1 methanol-acetone, and antigenretrieval can be conducted using one or more of: citrate buffer, SDS(detergent), and enzymatic treatment (e.g., trypsin, proteinase K).Additionally or alternatively, variations of the specific example caninclude combination of fluorescent markers with bright field staining(e.g., with methylene blue, with eosin, with DAPI) in order to mitigateinterference (e.g., spectral overlapping of fluorophores) producedduring “multi-color” staining. As such, distinguishing the subpopulationof cells can include transmitting excitation wavelengths of light tocaptured cells in the set of pores, and/or receiving emitted light fromfluorophores bound to the set of cells. However, in other variations,the reagents can include any other suitable reagents that distinguish atleast one subpopulation of cells from the set of cells (e.g., byenabling detection of any other suitable biomarker phenotype), thereagents can be delivered in any other suitable manner, using any othersuitable system, and any other suitable fixation, antigen retrieval,and/or staining protocol can be used.

Block S230 recites: encapsulating the set of cells and the set ofreagent particles within an encapsulation matrix at the set of pores,and functions to isolate captured cells of interest and reagents in asingle-cell format, in order to facilitate further processing andanalysis of the set of cells at a single-cell level. The encapsulationmatrix preferably isolates a pore and its contents within an array, inan embodiment of the system 100 described above; however, theencapsulation can isolate cells and reagent particles in any othermanner and/or in any other suitable system. The encapsulation matrixpreferably has a flow state and a set state, wherein a photochemicalreaction, thermochemical reaction, polymerization reaction and/or anyother suitable reaction switches the encapsulation matrix from the flowstate to the set state. In the flow state, the encapsulation matrix ispreferably substantially viscous, such that the encapsulation matrixdoes not flow into the pores during introduction into the system 100. Inthe set state, the encapsulation matrix is preferably a solid or gelthat prevents particle egress from the pores 111 (e.g., egress of cells,reagent particles, and large nucleic acid molecules from the pores), andis preferably porous or selectively permeable to permit small molecule,buffer, and reagent (e.g., detergent, enzyme, primer, etc.) penetrationtherethrough. Furthermore, by changing the constituents of a buffer orreagent and allowing sufficient time for diffusion, specificreagents/buffers can be entered into or eluted out from encapsulatedcells. In one variation, the encapsulation matrix is a microporousagarose gel with a low melting point, and in another variation, theencapsulation matrix is a photopolymerizable hydrogel, such as PEG orpolyacrylamide with photoinitiator; however, the encapsulation matrixcan alternatively be any suitable material with any other suitablepolymerization agent.

In a specific example of Block 230, implemented at an embodiment of thearray, the inlet channel, and the outlet channel of the system 200described above, the encapsulation matrix is a low melting agarose gelthat is received in its flow state at the first port of the inletchannel, and transmitted across the set of pores containing capturedcells and reagent particles, wherein excess encapsulation matrix istransmitted to the fourth port of the outlet channel to facilitate evendistribution of the encapsulation matrix. A portion of the encapsulationmatrix upstream of the pore channels is then replaced by a displacementfluid (e.g., air, immiscible fluid, oil) by transmission of thedisplacement fluid from the first port of the inlet channel to thesecond port of the inlet channel, thereby forming a displacement layer.The displacement layer facilitates diffusion of reagents and buffersacross the encapsulation matrix for further processing of the set ofcells. In the example, upon cooling of the agarose gel below its gelpoint, the cells and reagent particles are entrapped at the set of poresby the setting of the encapsulation matrix.

Block S240 recites: delivering a lysing reagent across the encapsulationmatrix, thereby lysing the set of cells, and functions to releaseintracellular content of the set of cells, which can be amplified andprocessed in order to individually analyze each cell of the set ofcells. The lysing reagent can additionally or alternatively includeprotein-digesting reagents (e.g., pepsin, proteinase K). In Block S240,lysing preferably includes delivering the lysing reagent to an interfaceof the encapsulation matrix (e.g., at a displacement layer generated bydelivering a displacement fluid through the inlet channel), such thatthe lysing agent can diffuse across the encapsulation matrix to a cellscaptured at the set of pores. The lysing reagent can be delivered at lowpressure to facilitate passive diffusion, or can be provided withpressure (e.g., positive pressure, negative pressure), in order to drivethe lysing reagent across the encapsulation matrix. In one variation,the lysing reagent comprises detergent and alkaline buffer that cantraverse across the porous encapsulation matrix. In a specific example,the lysing reagent comprises 5 microliters of 0.4 M KOH with 10 mM EDTAand 50 mM DTT, which is incubated with the set of cells at 65 C for 10minutes. In the specific example, lysis is subsequently terminated byadding 5 microliters of a neutralizing buffer including 0.9 M Tris-HClbuffer at pH 8.3, with 0.3 M KCl and 0.2 M HCl. However, in othervariations of Block S240, the lysing reagent can include any othersuitable lysing reagent, and/or lysis can be terminated in any othersuitable manner. In Block S240, lysing can further comprise heating theset of cells, the set of reagent particles, and the lysing reagent inorder to facilitate cell lysis. In variations, heating can be performedat a constant temperature or with a variable temperature profile. In aspecific example, implemented at an embodiment of the system 100described above, the lysing reagent can be received at the first portand transmitted to the second port of the inlet channel, and a region ofthe substrate proximal the set of pores can be heated with athermocycler (e.g., a block thermocycler comprising one or more heatingelements), in order to further enhance lysis. In Block S240, reagentproducts can further be removed post-lysis, by equilibrating contents ofcell sacs produced by lysis with a suitable buffer.

Block S250 recites: amplifying nucleic acid content of the set of cellsat the set of pores, thereby facilitating analysis of the set of cells,which functions to amplify genetic content in order to facilitatedownstream analyses of the set of cells at a single-cell level. In onevariation, amplifying nucleic acid content of the set of cells canfacilitate downstream analyses of the set of cells using electrophoreticassays; however, in other variations, amplifying nucleic acid content ofthe set of cells can facilitate any other suitable assay. In somevariations, downstream assays utilizing amplified nucleic acid contentof the set of cells can be implemented “on-chip” using an embodiment ofthe system 100 described above; however, in other variations, amplifiednucleic acid content can be retrieved from a system and analyzedoff-chip using any other suitable method.

In some variations, as shown in FIG. 12, Block S250 can includeperforming whole genome amplification (WGA) at the set of porescontaining lysed cells S252, which functions to expand a quantity ofnucleic acid content of a cell of the set of cells, in order tofacilitate downstream analyses of the set of cells requiring asufficient quantity of genetic content. In some variations, Block S252can function to provide a sufficient quantity of nucleic acids (e.g.,DNA, RNA) for further multiplex AS-PCR (e.g., as in Block S254) formutation analysis. In one variation, Block S252 can include performingWGA by multiple displacement amplification (MDA), which is a non-PCRbased DNA amplification technique wherein amplification can take placeat a constant temperature (e.g., 30 C). As such, in variations of BlockS252 implementing at least a portion of the system 100 described above,a region of the substrate proximal the set of pores can be isothermallyincubated (e.g., isothermally incubated in a thermocycler) in order todrive the WGA process to completion; however, in other variations, thelysed cells can be incubated in any other suitable manner. In a specificexample, bacteriophage φ29 DNA polymerase and randomexonuclease-resistant hexamer primers are used in an isothermal reactionat the set of pores for MDA. In the specific example, the bacteriophageφ29 DNA polymerase has high processivity, generating amplified fragmentsof >10 kb by strand displacement, and has proof-reading activityresulting in low misincorporation rates. In the specific example, amaster mix is prepared using REPL-g reaction buffer and REPL-g DNApolymerase with nuclease-free water, which is flowed into the inletchannel of an embodiment of the system 100 described above at the firstport. The substrate is then incubated at 30 C for 8-18 hours followed byheating of the substrate for 3 minutes at 65 C to inactivate the REPL-gDNA polymerase. In variations of the specific example, the yield ofamplified genetic content (e.g., DNA) can subsequently be measured usinga fluorochrome specific for double stranded DNA (e.g., SYBR green). Inother variations of Block S252, however, WGA can be performed in anyother suitable manner, such as a PCR-based technique for WGA (e.g.,degenerate oligonucleotide PCR, primer extension preamplification).

In some variations, as shown in FIG. 12, Block S250 can additionally oralternatively include performing AS-PCR at the set of pores containinglysed cells S254, which functions to enable detection of at least onemutation or other identifying feature characterizing cells of the set ofcells. In some variations, Block S254 can enable development ofmultiplex biomarker panels for detection of a type of breast cancer(e.g., biomarker panels for breast cancer); however, Block S254 canadditionally or alternatively enable development of any other suitablemarker profile for any other suitable cell-type of interest. Preferably,performing AS-PCR in Block S254 is based upon discrimination by Taqpolymerase between a match and a mismatch at the 3′ end of a PCR primer.In a specific example, Block S254 includes performing AS-PCR at anembodiment of the system 100 described above, wherein 25 microliters ofa PCR master mix including HotStartTaq, Type-it mutation detectionbuffer, dNTPs, and an equal concentration of primer (e.g., to a finalconcentration of 0.25 μM) is delivered into the first port of the inletchannel and allowed to diffuse across the encapsulation matrix to thelysed set of cells (e.g., the cell sacs) at the set of pores. PCRamplification in the specific example is then carried out using thefollowing thermocycling parameters: 95 C for five minutes for initialactivation, followed by 35 cycles of 95 C for 30 seconds, 6° C. for 90seconds, and 72 C for 30 seconds, followed by a final extension of 68 Cfor 10 minutes. In the example, conjugation of the forward primers onone end to the reagent particles delivered in Block S220 allowslocalization of amplified amplicons at the set of pores.

In the specific example of Block S254 described above, and variationsthereof, primer pairs for AS-PCR to detect single nucleotidepolymorphism (SNP) mutations for cell biomarkers (e.g., breast cancerbiomarkers) can be used, as shown in FIG. 13. In the specific example,for each SNP mutation, two AS forward primers and a reverse commonprimer are preferably required. Furthermore, a tail is incorporated inthe AS primers, thus allowing differentiation of the alleles through thelength of the PCR amplicon on the encapsulation matrix (e.g., agarosegel). In one example of design of forward primers for AS-PCR, a forwardprimer can be designed without a tail, and a 5-base pair short tail canbe added to the 5′ end of a wild type forward primer, while a 15-basepair long tail can be added to the 5′ end of a mutant forward primer,which allows discrimination of 10-base pairs to be detected between twoAS-PCR amplicons. In the example, the melting temperature can beconfigured to be between 50 C and 65 C, with no more than 5 C differencebetween melting temperatures for the wild type forward primer, themutant forward primer, and a common reverse primer. Furthermore, in thespecific example, to multiplex AS-PCR for detecting multiple mutations(e.g., 5 mutations) simultaneously, multiple mutations (e.g., fivebreast cancer mutations) with different amplicon sizes differing by atleast 20 base pairs can be chosen. However, in other variations andexamples, any other suitable forward primers with any other suitablenumber of tail base pairs for wild type and/or mutant primers can bechosen, any other suitable reverse primers can be chosen, primers can bechosen with any other suitable melting temperature, the multiplex AS-PCRcan be configured to detect any other suitable number of mutations, andthe mutations can be characterized by any other suitable amplicon size(e.g., number of base pairs). Furthermore, In other variations of BlockS254, performing AS-PCR can alternatively be based upon discriminationusing any other suitable master mix incorporating any other suitablepolymerase(s), discrimination between a match and a mismatch at anyother suitable location of a genetic sequence, and/or any other suitablethermocycling profile.

In variations wherein reagent beads for WGA and AS-PCR are co-deliveredin Block S220, to prevent interference from effects of forward primersfor AS-PCR on the WGA process, the forward primers for AS-PCR in BlockS254 can be modified with one or more thermolabile 4-oxo-1-pentyl (OXP)phosphotriester (PTE) modification groups at 3′-terminal and3′penultimate inter nucleotide linkages. The OXP PTE modifications canthus impair polymerase primer extension at the lower temperatures thatexist prior to PCR amplification in Block S254. Interference from theforward primers can, however, be mitigated using any other suitablemodification groups, and/or in another suitable manner.

In some variations, as shown in FIG. 12, Block S250 can additionally oralternatively include performing RT-PCR for a subpopulation of the setof cells S256, in order to facilitate comparisons of gene expression fora subpopulation of the set of cells. Block S256 can be performed at theset of pores before or after encapsulation in Block S230, or canadditionally or alternatively be performed after cell retrieval (e.g.,in variations of the method 200 incorporating Block S280). In onevariation, Block S256 can include performing qRT-PCR on a subpopulationof captured CSCs in order to compare their gene expression with othercancer cells and leukocytes (e.g., from a biological sample comprising avolume of blood). In a specific example of this variation, thesubpopulation of CSCs can be incubated (e.g., on-chip, off-chip) with areverse transcription and pre-amplification master mix (e.g., CellDirectOne-step qRT-PCR kit), with SUPERase® RNAse inhibitor.Post-amplification in the specific example, threshold cycle values for agroup of target genes (e.g., Her2, ALDH1, TWIST1) and an internalcontrol (e.g., 18S rRNA) can be determined and recorded and relativequantitation of gene expression can be calculated using comparative CT(ΔΔET) and/or any other suitable method. However, Block S256 canalternatively include performing any other suitable type of PCR for anyother suitable subpopulation of the set of cells.

As shown in FIG. 10A, the method 200 can further include Block S260,which recites: transmitting a sieving matrix to a set of electrophoresischannels fluidly coupled to the set of pores. Block S260 functions toprovide a porous matrix that is continuous between the set of pores andthroughout the set of electrophoresis channels, in order to form acontinuous path for electrophoretic separation of amplifiedintracellular content. The sieving matrix is preferably similar to oridentical in composition to the encapsulation matrix delivered in BlockS230, and in a specific example, comprises 3% agarose. However, thesieving matrix can alternatively include any other suitable materialthat facilitates electrophoretic separation in Block S270. Additionally,the sieving matrix preferably matrix preferably has a flow state and aset state, wherein a photochemical reaction, thermochemical reaction,polymerization reaction or any other suitable reaction switches thesieving matrix from the flow state to the set state. In the flow state,the sieving matrix can thus be delivered to the set of electrophoresischambers and evenly distributed across them, and in the set state, thesieving matrix is preferably a solid or gel that is preferably porous orselectively permeable to permit small molecule, buffer, and reagentpenetration therethrough. In a specific example of Block S260implementing an embodiment of the system 100 described above, thesieving matrix is received under pressure at the third port of theoutlet channel and excess sieving matrix is passed out of the sixth portof the electrophoresis outlet channel. Subsequent to sieving matrixdelivery, separation buffer (e.g., 1×TBE buffer with 0.5 μg/mL ethidiumbromide) is then received at the first port, the second port, the thirdport, the fourth port, the fifth port, and the sixth port of thesubstrate and diffused across the encapsulation matrix/sieving matrix.The separation buffer can include fluorescence dye that can facilitateidentification of the size and location of a separated amplicon, and/orany other suitable component that facilitates identification of specificamplicons (e.g., in bands produced by electrophoresis). However, inother variations, the sieving matrix can be delivered in any othersuitable manner (e.g., by positive pressure, by negative pressure),transitioned to a set state in any other suitable manner, and/ordelivered with a separation buffer comprising any other suitablefactors.

Also shown in FIG. 10A, the method 200 can further include Block S270,which recites: transmitting an electric field across the substrate,thereby enabling electrophoretic analysis of the set of cells. BlockS270 functions to provide a driving force that electrokineticallyseparates amplified intracellular content from the set of cells, basedupon size and charge of the content. In some variations, Block S270 caninclude heating the substrate, which can facilitate release of amplifiedproducts from reagent particles (e.g., primer beads); however,variations of Block S270 can entirely omit heating the substrate, and/orcan include facilitating release of amplified products in any othersuitable manner (e.g., by pH shift). Preferably, transmitting theelectric field across the substrate includes applying a substantiallylarge electric field (e.g., a few kV/centimeter) across electrodescoupled to the substrate, using a voltage regulator. In variationsimplemented at an embodiment of the system 100 described above, theelectric field is preferably provided at the set of electrodes coupledat the substrate proximal the inlet channel and the electrophoresisoutlet channel; however, the electric field can alternatively beprovided in any other suitable manner at any other suitable apparatusconfigured to generate an electric field that provides a suitable forcefor electrokinetic separation. Bands produced by electrophoreticseparation can subsequently be viewed under fluorescence microscopy fordetection of the intensity and relative location of bands, and/or in anyother suitable manner for electrophoretic analysis of the set of cells.As such, the method 200 can enable distinguishing of amplicons basedupon size and color (i.e., by fluorophores), in order to facilitateexamination of multiple biomarkers in at least two different manners.

In some variations, as shown in FIG. 10A, the method 200 canadditionally or alternatively include Block S280, which recites:extracting at least one of a captured cell of the set of cells,intracellular content of a cell of the set of cells, and amplifiedintracellular content of a cell of the set of cells. Block S280functions to extract a cell of interest and/or intracellular content ofa cell of interest from the substrate, in order to facilitate furtheranalyses of a cell of the set of cells in a single-cell format. Invariations of Block S280 including extracting a captured cell of the setof cells, the captured cell is preferably extracted individually from apore of the set of pores; however, in some variations multiple cells ofthe set of cells can be extracted at the set of pores simultaneously. Inone variation, implementing an embodiment of the system 100 describedabove, a cell removal tool can be used to extract at least one capturedcell (e.g., a CSC, a contaminating leukocyte, a CTC, etc.), wherein thecell removal tool is configured to penetrate the inlet channel (e.g., atone or more of the first port and the second port), and facilitateextraction of a captured cell directly from a pore. In one example, thecell can be aspirated into the cell removal tool, and in anotherexample, the cell can be pushed into the cell removal tool (e.g., byproviding a positive pressure at the outlet channel). In still othervariations, however, the captured cell can be extracted in any othersuitable manner.

In variations of Block S280 including extracting intracellular contentof a cell of the set of cells, the intracellular content/cell sacs oflysed cells of the set of cells can be extracted by accessing thepore(s) of the set of pores containing the lysed cellular content, priorto amplification of intracellular content. In one variation, theintracellular content can be extracted by harvesting encapsulationmatrix of at least one pore. In a specific example implemented at anembodiment of the system 100 described above, the encapsulation matrixof a pore can be excised (e.g., by incision of a PMMA/COP laminate) toextract the intracellular content. However, in other variations, theintracellular content can be extracted in any other suitable manner forany other suitable downstream application. In variations of Block S280including extracting amplified intracellular content of a cell of theset of cells, amplified intracellular content can be extracted byharvesting encapsulation matrix containing the amplicons generated invariations of Block S250. In specific examples of these variations,implemented at an embodiment of the system 100 described above, theencapsulation matrix with amplicons can be excised (e.g., by incision ofa PMMA/COP laminate) from each pore of the set of pores, in order tofacilitate downstream assays performed “off-chip” (e.g., off-chipelectrophoresis). However, the amplified intracellular content can beextracted in any other suitable manner, and/or for any other suitabledownstream application.

In some variations, the method 200 can additionally or alternativelyinclude Block S290, which recites labeling the set of cells, in order todetermine a measure of efficiency. Block S290 functions to enablemeasurement of an efficiency of cell capture by a system 100 forcapturing and analyzing cells in a single-cell format, which can be usedto improve efficiency in the system and/or to identify causes ofinefficiencies in the system. Block S290 can be implemented prior toreception of the set of cells in Block S210, simultaneously withreception of the set of cells in Block S210, and/or in any othersuitable manner. Post capture at the set of pores, the labeled cells canbe imaged using fluorescence microscopy and/or any other suitableoptical detection module (or other module) in order to discriminatecaptured cells of interest from captured contaminants. In one variation,labeling can include labeling the cells with Cell Tracker green dye,which, in a specific example, includes centrifuging the set of cells at1000 rpm for 5 minutes, removing a supernatant, and adding 6 millilitersof serum-free media and 5 microliters of Cell Tracker dye to thecentrifuged cells, with incubation at 37 C for 30 minutes. In thespecific example, the dyed cells can then be centrifuged at 1000 for 5minutes with subsequent supernatant removal, washed in saline (e.g.,1×PBS), and resuspended in 10 milliliters of complete growth medium. Inanother variation, labeling in Block S290 can include antibody stainingof the set of cells. In a specific example, antibody staining can beimplemented “on-chip” using an embodiment of the system 100 describedabove, wherein prior to receiving a biological sample, surfaces offluidic channels of the substrate are coated by running 8 mL of ixPBS/1% BSA/2 mM EDTA buffer for 10 minutes in order to prevent celladhesion and bubble trapping. In the specific example, buffer (e.g., 1.5mL of PBS/BSA/EDTA) can be added to dilute a fixative solution mixedwith the biological sample, and the biological sample can be received byway of a pump configured to provide 6 kPA of pressure. In the specificexample, the captured cells are washed with 3 mL of wash buffer (e.g.,PBS/BSA/EDTA) and incubated with 2 mL of 4% formalin/i % BSA/0.1% Tritonfor 10 minutes, which is followed by another wash with 2 mL of washbuffer. The captured cells are then incubated with 4 mL of 5% goat serumfor 20 minutes, after which the goat serum is replaced with 1 mL ofprimary antibody cocktail comprising 1:200 CAM5.2, 1:400 CD45, and1:1000 Hoechst stain and incubated for 45 minutes. In the specificexample, the stained captured cells are then washed with 2 mL of washbuffer, incubated with 2 mL of secondary antibody cocktail (e.g., 3micrograms/mL of Alexa 488, 3 micrograms/mL of Alexa 568) for 30minutes, and then washed again with 2 mL of wash buffer. The stainedcaptured cells are then observed under fluorescence microscopy in orderto discriminate cells of interest from contaminants. However, labelingin Block S290 can include any other suitable type of labeling thatallows for discrimination between captured cells of interest andcontaminants.

2.1. Method—Example Application Areas

The method 200 described above can be used for a variety of biologicalassays and procedures. Running an assay or procedure preferably includescapturing and isolating target cells in addressable locations within thesystem and delivering reagents to the interior or surface of eachcaptured cell while maintaining cell registration with its respectivepore or location. Post-delivery of reagents, the captured target cellsand/or intracellular content produced by cell-lysis can either beprocessed and analyzed on-chip, or can be harvested for processing andanalysis off-chip. Cell analysis is preferably used to determine themorphology of the captured cells, to examine additional phenotypicexpressions of the captured cells (e.g., by biomarker characterization),and to determine the number and location of captured cells of interest.Cell analysis is preferably performed by an associated integratedplatform 30, wherein morphology, biomarker expression (e.g., as examinedunder fluorescence), and cell counting are preferably accomplishedthrough global chip imaging and image analysis. Imaging and analysis ispreferably automatically performed, but can alternatively besemi-automated or manually performed. However, morphology determination,biomarker expression, and cell counting can be achieved through anyother suitable method.

Running assays on the isolated cells functions to determinecharacteristics of the cells and/or determine cell responses to givenstimuli. Analyses can be run on the cells individually (e.g. single celllevel analysis), wherein cells can be individually fluidly isolatedwithin the system 100. Alternatively, analyses can be run on the system100 as a whole. Example assays that can be run on the cells include FISHassays, mRNA FISH assays, ISH assays, selective cell lysing and lysatecollection, single cell molecular analysis (e.g. PCR, RT-PCR, WholeGenome Amplification, ELISPOT, ELISA, Immuno-PCR, etc.), drug testing,cell culturing, affinity analyses, time-responsive analyses, but otheranalyses can alternatively/additionally be run. Isolated cells can beremoved prior to, during, or after the assays have been run, preferablywith the cell removal tool 600 but alternatively with any suitablemethod. Alternatively, isolated cells can be isolated within the chamber113 (e.g. with an isolation layer), fixed, cultured within the chamber113, or be retained within the chamber 113 in any other suitable manner.

In one specific application, as shown in FIG. 14A, the method 200 can beused to isolate CTCs from a biological sample, process the CTCs on-chipfor WGA and AS-PCR, and analyze the CTCs on-chip by electrophoreticseparation and fluorescent detection in order to characterize the set ofCTCs. In another specific application, as shown in FIG. 14B, the method200 can be used to identify and isolate a subpopulation of CSCs from aset of CTCs, wherein the CSCs can be retrieved and analyzed by usingqRT-PCR to characterize gene expression of isolated CSCs. However, inother specific applications, the method 200 can be used to process andanalyze any other suitable set of cells/subpopulation of the set ofcells, using any other suitable assay.

3. Integrated Platform

As shown in FIG. 15, the system 100 and/or method 200 can be implementedwith an integrated platform 30 including a sample workstation 40 and animaging platform 50. The integrated platform 30 is preferably fullyautomated, but can alternatively be semi-automatic or manually operated.The integrated platform 30 can perform all or some the functions ofpipetting, aliquoting, mixing, pumping, thermal incubation,theromocycling, monitoring, and analysis (e.g., by fluorescentdetection). The integrated platform 30 can additionally automaticallyidentify occupied chambers 113, image said chambers 113, and/or performanalyses on said chambers 113. The integrated platform 30 canadditionally selectively remove cells from the system 100. Invariations, the integrated platform 30 can include an embodiment of anintegrated platform 50 as described in U.S. Pub. No. 2013/0190212,entitled “Cell Capture System and Method of Use” filed 25 Jul. 2012,which is incorporated herein in its entirety by this reference. However,the integrated platform 30 can be any other suitable integrated platform30, and can additionally or alternatively perform any other suitablefunction. The integrated platform 30 is preferably utilized with asystem 100 as described above, but can alternatively be utilized withany suitable system 100 or method 200.

The system 100 and method 200 of the preferred embodiment and variationsthereof can be embodied and/or implemented at least in part as a machineconfigured to receive a computer-readable medium storingcomputer-readable instructions. The instructions are preferably executedby computer-executable components preferably integrated with the systemand one or more portions of a processor and/or a controller. Thecomputer-readable medium can be stored on any suitable computer-readablemedia such as RAMs, ROMs, flash memory, EEPROMs, optical devices (CD orDVD), hard drives, floppy drives, or any suitable device. Thecomputer-executable component is preferably a general or applicationspecific processor, but any suitable dedicated hardware orhardware/firmware combination device can alternatively or additionallyexecute the instructions.

The FIGURES illustrate the architecture, functionality and operation ofpossible implementations of systems, methods and computer programproducts according to preferred embodiments, example configurations, andvariations thereof. In this regard, each block in the flowchart or blockdiagrams may represent a module, segment, or portion of code, whichcomprises one or more executable instructions for implementing thespecified logical function(s). It should also be noted that, in somealternative implementations, the functions noted in the block can occurout of the order noted in the FIGURES. For example, two blocks shown insuccession may, in fact, be executed substantially concurrently, or theblocks may sometimes be executed in the reverse order, depending uponthe functionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts, or combinations of special purpose hardware andcomputer instructions.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

We claim:
 1. A method for capturing and analyzing a set of cells,comprising: capturing the set of cells, including a subpopulation ofcells, at a set of pores of a substrate, each pore 111 the set poresincluding a chamber operable to retain a single cell of the set of cellsand comprising: an inlet, an outlet, and a set of walls defining achamber volume directly corresponding to the volume of the single cellof the set of cells; transmitting a reagent volume to the set of poresthrough a manifold fluidly coupled to the set of pores, wherein thereagent volume is configured to distinguish the subpopulation of cellsfrom the set of cells; guiding a cell removal tool to a pore of the setof pores containing a cell of the subpopulation of cells; and receivingthe cell of the subpopulation of cells from the pore and into the cellremoval tool.
 2. The method of claim 1, further including: transmittingexcitation wavelengths of light into each pore of the set of pores aftertransmission of the reagent volume, thereby enabling identification ofat least one cell of the subpopulation of cells.
 3. The method of claim2, further comprising transmitting a permeabilization reagent, throughthe manifold, to the set of pores prior to transmission of the reagentvolume, wherein the reagent volume comprises fluorescent markers.
 4. Themethod of claim 3, further comprising: at the capture substrate,performing a fluorescence in situ hybridization (FISH) assay on the setof cells, thereby evaluating cancer-associated cells of the set of cellsin single-cell format.
 5. The method of claim 4, wherein performing thefluorescence in situ hybridization (FISH) assay comprises performing anmRNA FISH assay.
 6. The method of claim 2, wherein capturing the set ofcells comprises capturing a set of circulating tumor cells (CTCs), andwherein the subpopulation of cells includes a subpopulation ofcirculating stem cells (CSCs).
 7. The method of claim 6, whereintransmitting excitation wavelengths of light further includes enablingidentification of a CD44⁺/CD24⁻ phenotype characterizing cells of thesubpopulation of CSCs.
 8. The method of claim 7, further comprising:upon receiving the cell of the subpopulation of cells, performingpolymerase chain reaction (PCR) with nucleic acid content of the cell ofthe subpopulation of cells, wherein performing PCR includes performingPCR for a CSC of the set of cells, and determining threshold cyclevalues for a group of target genes including at least one of Her2,ALDH1, and TWIST1.
 9. The method of claim 1, wherein transmitting thereagent volume includes transmitting an antibody cocktail including CD24and CD44 antibodies, and, wherein transmitting the antibody cocktailfurther includes transmitting an additional antibody to the set ofpores, wherein the additional antibody enables identification ofcontaminating cells captured at the set of pores.
 10. The method of 1,further comprising culturing the subpopulation of cells at the set ofpores of the substrate, and performing a live cell assay on thesubpopulation of cells, within the set of pores of the substrate.
 11. Amethod for capturing and analyzing a set of cells, comprising: capturingthe set of cells, including a subpopulation of cells, at a set of poresof a substrate, each pore 111 the set pores including a chamber operableto retain a single cell of the set of cells and comprising: an inlet, anoutlet, and a set of walls defining a chamber volume directlycorresponding to the volume of the single cell of the set of cells; witha fluid delivery system coupled to the substrate, transmitting a reagentvolume to the set of pores through a manifold fluidly coupled to the setof pores, wherein the reagent volume is configured to distinguish thesubpopulation of cells from the set of cells; culturing thesubpopulation of cells at the set of pores of the substrate, andperforming a live cell assay on the subpopulation of cells, within theset of pores of the substrate; and with an automated system, guiding acell removal tool to a pore of the set of pores containing a cell of thesubpopulation of cells, and receiving the cell of the subpopulation ofcells from the pore and into the cell removal tool.
 12. The method ofclaim 11, wherein transmitting a reagent volume to the set of porescomprises performing an optical detection-based assay upon thesubpopulation of the set of cells.
 13. The method of claim 12, furthercomprising: transmitting a permeabilization reagent, through themanifold, to the set of pores prior to transmission of the reagentvolume, wherein the reagent volume comprises fluorescent markers; andperforming a fluorescence in situ hybridization (FISH) assay on the setof cells at the substrate, thereby evaluating cancer-associated cells ofthe set of cells in single-cell format.
 14. The method of claim 13,wherein performing the fluorescence in situ hybridization (FISH) assaycomprises performing an mRNA FISH assay.
 15. The method of claim 12,wherein the reagent volume comprises an antibody cocktail comprisingCD24 and CD44 antibodies, and wherein the method further comprisestransmitting a bright field stain with the antibody cocktail, therebymitigating interference due to spectral overlapping of fluorophores. 16.The method of claim 12, wherein the reagent volume comprises a lysisreagent, the method further comprising performing polymerase chainreaction (PCR) with nucleic acid content of at least one of thesubpopulation of cells at the substrate, wherein performing PCR includesperforming PCR for a CSC of the set of cells.
 17. The method of claim16, wherein performing PCR comprises performing whole genomeamplification for the CSC at the substrate, and upon removal of the CSCfrom the substrate with the cell removal tool, detecting singlenucleotide polymorphisms associated with the CSC upon performingallele-specific PCR (AS-PCR) with amplified nucleic acid material of theCSC.
 18. The method of claim 12, further including: transmittingexcitation wavelengths of light into each pore of the set of pores aftertransmission of the reagent volume, thereby enabling identification ofat least one cell of the subpopulation of cells, wherein transmittingexcitation wavelengths of light further includes enabling identificationof a CD44⁺/CD24⁻ phenotype characterizing cells of the subpopulation ofcells.
 19. The method of claim 11, wherein receiving the cell of thesubpopulation of cells from the pore and into the cell removal toolcomprises receiving, by capillary force, the cell into the cell removaltool.
 20. The method of claim 19, wherein receiving the cell of thesubpopulation of cells from the pore and into the cell removal toolcomprises receiving the cell upon delivery of positive pressure throughthe outlet of at least one pore of the set of pores.